EP3517606B1 - Verfahren zur herstellung eines supplements aus mesenchymalen zellkulturen von wharton-sulze und verwendungen davon - Google Patents

Verfahren zur herstellung eines supplements aus mesenchymalen zellkulturen von wharton-sulze und verwendungen davon

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Publication number
EP3517606B1
EP3517606B1 EP17853510.0A EP17853510A EP3517606B1 EP 3517606 B1 EP3517606 B1 EP 3517606B1 EP 17853510 A EP17853510 A EP 17853510A EP 3517606 B1 EP3517606 B1 EP 3517606B1
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European Patent Office
Prior art keywords
supplement
cells
fibroblasts
equivalent
culture medium
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Active
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EP17853510.0A
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English (en)
French (fr)
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EP3517606A1 (de
EP3517606A4 (de
EP3517606C0 (de
Inventor
Andrés Eliú CASTELL RODRÍGUEZ
Miguel Ángel HERRERA ENRÍQUEZ
Gabriela PIÑÓN ZÁRATE
Katia JARQUÍN YÁÑEZ
Casandra CHAIRES ROSAS
Rosa María ARELLANO OLIVARES
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Universidad Nacional Autonoma de Mexico
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Universidad Nacional Autonoma de Mexico
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Publication of EP3517606A1 publication Critical patent/EP3517606A1/de
Publication of EP3517606A4 publication Critical patent/EP3517606A4/de
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Publication of EP3517606C0 publication Critical patent/EP3517606C0/de
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0629—Keratinocytes; Whole skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/33—Fibroblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/362—Skin, e.g. dermal papillae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
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    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • A—HUMAN NECESSITIES
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    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • C12N5/0698—Skin equivalents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/64—Animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/02—Coculture with; Conditioned medium produced by embryonic cells
    • C12N2502/025—Coculture with; Conditioned medium produced by embryonic cells extra-embryonic cells, e.g. amniotic epithelium, placental cells, Wharton's jelly

Definitions

  • the present invention pertains to the field of tissue engineering.
  • the present invention concern to a method for preparing a supplement from mesenchymal cell cultures of Wharton's jelly and uses of same.
  • the cutaneous trauma cause diverse conditions that put at risk the health of patient, between these conditions are included dehydration, loss of electrolytes and proteins in the wound site, at the same time that the wound remains exposed to bacterial infections.
  • dehydration loss of electrolytes and proteins in the wound site, at the same time that the wound remains exposed to bacterial infections.
  • diverse methods has been developed, allowing replacement of the main components of skin, epidermis, and dermis, with biological equivalents produced in vitro.
  • cells 3T3 have been used to induce the proliferation of keratinocytes in culture.
  • the cells 3T3 are mouse embrionary fibroblasts, serving as feeder cells (humans or animals) or "nurses" ( Rheinwald JG, Green H., Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells, 1975 ). This method has been widely used to grow keratinocytes.
  • the previous method comprises to grow the obtained cells from a biopsy in presence of irradiated cells 3T3, subsequently; the culture of keratinocytes washes and collects.
  • the previous methodology has allowed to obtain large quantities of keratinocytes, in order to construct epidermal equivalents including cutaneous equivalents or dermal equivalents used to treat patients with second or third degree burns ( Llames S, Garcia E, Garcia V, del Rio M, Larcher F, Jorcano JL, et al. Clinical results of an autologous engineered skin, 2006 ).
  • mesenchymal cells applied directly in wounds promotes the recovery by an indirect mechanism, delivering diverse molecules that may have a therapeutic effect, for example, growth factors and other cytokines, while the mesenchymal cells can differentiate to fibroblasts ( Chui-Yee Fong, Human Wharton's Jelly Stem Cells and Its Conditioned Medium Enhace healing of Excisional and Diabetic Wounds, 2014 ).
  • the mesenchymal cells obtained from Wharton's jelly secrete different factors, which can induce the proliferation of keratinocytes and fibroblasts in culture.
  • the supplement obtained by the method of present invention can be used as additive in the cell cultures from cutaneous system, to elaborate a cell suspension of fibroblasts and to use in the treatment of diseases, defects, cutaneous disorders as result from cutaneous aging.
  • the application of cultivated fibroblasts using the supplement obtained by the method developed increases the cellular activation, promoting an increase from mechanical stress in the extracellular matrix, inducing an elongation from fibroblasts and the stimulation in the collagen synthesis, release of growth factors, inhibition from metalloproteinase secretion, and increasing the secretion from inhibitor factors of the same.
  • the supplement is prepared from the supernatant of mesenchymal cell cultures obtained from Wharton's jelly cultivated in specific conditions.
  • the supplement includes growth factors necessary to favor and increases the cellular proliferation in vitro for several cells from cutaneous system.
  • the present invention is related with the production of a supplement from mesenchymal cell cultures of Wharton's jelly and its use in the development of cell cultures from cutaneous system in vitro.
  • the cells of cutaneous system comprise fibroblasts, keratinocytes, dendritic cells, monocytes or combinations of the same, which can be cultivated in an isolated manner.
  • the supplement obtained by the method in the present invention can be used in the expansion of cutaneous cell populations or incorporated in culture means for the same, independently from intended use of the cells.
  • the present invention provides a method to obtain a supplement from mesenchymal cells of Wharton's jelly.
  • the method to obtain the supplement comprises:
  • the mesenchymal cells of Wharton's jelly used in the present invention are obtained from collect a fragment of umbilical cord, under aseptic conditions, subsequently the size of umbilical cord is reduced by a medical and enzymatic treatment.
  • “Cultivate mesenchymal cells of Wharton's jelly” refers to cultivate treated fragments from umbilical cord in a suitable culture mean to reach a suitable confluence.
  • “Harvest the cultivated cells” refers to separate the previous cultivated cells from culture mean by an enzymatic treatment; wash the previous cultivated cells; and seeds the previous cultivated cells in a new suitable culture mean during a determined time, preferably 48 hours.
  • “harvest the cultivated cells” is equivalent to "step”.
  • the cell culture in accordance with the steps a) and b), is repeated preferably at least once, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times.
  • the conditioned mean refers to culture mean obtained from seed or steps, that is found free of cells. Besides contain the proper components of their formulation, contains the secretion products (proteins, hormones, development factors, cytokines, and signaling molecules) that are produced and delivered by the cells in question during its culture.
  • the term "supplement” corresponds to the conditioned mean by the mesenchymal cells concentrated and sterilized.
  • the supplement can be used in a culture mean.
  • the supplement from present invention comprises several cytokines, for example, the compounds Actine, Annexin A1, Amyloid beta A4, ATP synthase sub-unity-B, Cadherin 2, CD44, Collagen chain alpha-1(VIII), EGF, derived factor of epithelium pigmentary, Fibronectin, FGF-2, Inhibin chain Beta, Integrin B1, Interleukin 6, ILGF-bp4, ILGF-bp7, Keratin 2, Laminar sub-unity Alpha-2, Lipoprotein lipase, MAPK1, Myosin 9, Moesin, M-CSF, MPI-1, PI-2, Nexin derived of glia, NDP-kA, NDP-kB, PAI-1, PDGF sub-unity A, Peroxyredoxin 1, Peroxyredoxin 2, Complementary protein 5 of reparation to XR , protein ih-h3 induced by TGFB, Purine nucleoside phosphorilase, S
  • meenchymal cells of Wharton's jelly refers to multi-potential cells from mesoderm obtained from umbilical cord.
  • confluence refers to the number of cells in a cell culture and is referred to grade in which the cells cover the surface of a solid culture mean or volume of a cellular culture. The confluence is measured in % with respect to occupied total volume.
  • suitable confluence refers to a suitable percentage (%) to be used in a process, for example, in the elaboration from supplement, elaboration from dermal equivalent or cutaneous equivalent in accordance with the present invention.
  • a suitable percentage % to be used in a process, for example, in the elaboration from supplement, elaboration from dermal equivalent or cutaneous equivalent in accordance with the present invention.
  • such percentage is greater than 60%, more preferably, is greater than 80%.
  • suitable culture mean refers to a culture mean for mesenchymal cells, for example, a DMEM-F12 mean (DMEM 50% - F12 50%) supplemented with 10% bovine fetal serum 10% and antibiotics.
  • the suitable culture mean is supplemented with development factors, namely epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF).
  • EGF epidermal growth factor
  • bFGF basic fibroblast growth factor
  • the present invention provides methods to obtain an epidermal equivalent using the supplement from present invention.
  • the epidermal equivalents refer to epithelial cells laminates grown over a culture.
  • the epidermal equivalents are useful to be applied in patients with second degree superficial burns or with cutaneous chronic ulcers of different etiology.
  • the epidermal equivalents can be dermal or cutaneous equivalents.
  • the term "dermal equivalent” refers to a tissue construed by tissue engineering comprising a cellular layer of fibroblasts and an extracellular matrix organized similarly to dermal tissue in the skin.
  • the fibroblasts are located placed within a scaffold of natural, synthetic or composite biomaterials.
  • the term "cutaneous equivalent” refers to a tissue constructed by tissue engineering comprising an internal layer and an external layer, in which the internal layer comprises the dermal complex, and the external layer comprises the epidermal complex constituted by a monolayer of keratinocytes. Both layers are organized similarly to dermis and epidermis in the skin, respectively. In a modality, the external layer consists of stratified plane epithelium with stratum corneum. The epidermal equivalent is located placed over the dermal equivalent constructed with natural, synthetic or composite biomaterials.
  • the method to obtain the dermal equivalent comprises:
  • the fibroblasts used in the method to obtain the dermal equivalent can be obtained from autologous or allogenic cells from cutaneous system, from which the fibroblasts of dermis are separated by an enzymatic treatment.
  • cultivate fibroblasts refers to maintain fibroblasts in a culture mean comprising the supplement from present invention to reach a suitable confluence; separate previous cultivated cells from culture mean by an enzymatic treatment; wash the previous cultivated cells; and reseeds the cells in a culture mean comprising the supplement from present invention to reach a suitable confluence for the construction from dermic equivalent.
  • the supplement from present invention is used in a concentration from of 5% to 50% with respect to total volume form culture mean.
  • Produce the dermic equivalent refers to place the fibroblasts cultivated previously in a solution containing fibrinogen and prothrombin in presence of antifibrinolytics, sodium chloride and calcium chloride, to form a cellular suspension; place the cellular suspension in a container and incubate to its gelation; and place the gel over a scaffold of natural, synthetic or composite biomaterials with adhesive biocompatible.
  • the method to obtain the cutaneous equivalent comprises:
  • cultivate keratinocytes refers to cultivate keratinocytes in a culture mean comprising the supplement from present invention to reach a suitable confluence; separate previous cultivated cells from culture mean by an enzymatic treatment; wash the previous cultivated cells; and reseeds the cells in a culture mean comprising the supplement from present invention to reach a suitable confluence, necessary to the construction the cutaneous equivalent.
  • the supplement from present invention is used in a concentration of 5% al 50% with respect to total volume from culture mean.
  • Produce the cutaneous equivalent refers to seed and cultivate the keratinocytes previously cultivated over gelled fibroblasts in a culture mean added with the supplement from present invention to reach the suitable confluence; and place over a scaffold of natural, synthetic or composite biomaterials with adhesive biocompatible.
  • the fibroblasts used to elaborate the cutaneous equivalent are obtained according to the elaboration method of dermic equivalent.
  • the supplement from present invention is used in a concentration of 5% to 50% with respect to total volume from culture mean.
  • autologous system comprises an epithelial and dermal cells system obtained from the same person to which the tissue will be grafted.
  • allogenic system comprises epithelial and dermic cells obtained from a different person to which the tissue will be grafted.
  • Dermal equivalents obtained by the method of the present invention may be used to treat diseases, defects, dermal disorders, or wounds.
  • the dermal equivalents from present invention are useful to induce in the patient a regeneration from conjunctive tissue, to subsequently promote the reepithelialization.
  • the use of dermal equivalents from present invention consists in apply directly the dermal equivalent over the wound bed at least once a week to regenerate the dermal and epidermal tissues from patient, what is achieved un a period from 2 to 14 weeks treatment, preferably 4 to 8 weeks.
  • the candidates to receive the treatment with dermal equivalents comprise patients presenting diseases, defects, dermal disorders, or wounds, for example, ulcers, chronic wounds, vascular, arterial, venous, and lymphatic, of decubitus or diabetic foot.
  • the patient must be free of infection in wound and in case of being diabetic with the controlled glycaemia.
  • the wound must be washing by the use of antiseptic solution to subsequently debridement from lesion.
  • the dermal equivalent is directly implanted over the injured tissue, subsequently is covered with an insulating bandages to prevent its detachment, keeping in that way by a sufficient time to reach the closure of the wound.
  • Cutaneous equivalents obtained by the method of the present invention may also be used in the treatment from diseases, dermal defects, cutaneous disorders or wounds when the thickness from condition is partial or with an extension requiring graft.
  • the use from cutaneous equivalent is applicable for example, when the dermal defect is a nevus, tattoo, hypertrophic scar, keloid or burn scar or of a size large enough to be surgically removed.
  • the use from cutaneous equivalent can be applied for the case of second or third degree burns from a size large enough to be surgically removed.
  • the implantation from cutaneous equivalent is performed by a surgical procedure, in which the cutaneous equivalent is placed as a graft immediately after of retirement from affected tissue, fixing by using sutures, surgical staples or a suitable adhesive, to subsequently cover with un an insulating bandage to avoid its detachment.
  • the affected zone remains covered by a sufficient time to reach the closure of the wound.
  • a method to manufacture a cellular suspension of fibroblasts comprises:
  • the obtained supplement from developed method or cellular suspension of fibroblasts described in the above paragraph can be used to favor the growth and activation in vitro from cells of cutaneous system which can be inactive by age, for example in the skin of adult patients with fine, moderate to deep, or deep wrinkles; loss of dermal tone and subcutaneous tissue; injuries in the skin such as atrophic scares as acne marks grade I (macular), II (mild), III (moderate), and IV (serious) and injuries of cutaneous caused by red or white stretch marks; pigmentary injuries from type solar or senil lentigo and simple lentigo, melasma and melanodermias from different etiology; injuries of acne; photo-aging severe classified with Fiztpatrick's scale from I, II, or III, in Glogau's scale fom 1, 2, 3, or 4 and in SCINEXA's scale from 0, 1, 2, or 3 characterized in that loss of elasticity and tissue tone, this because fibroblasts gradually losing
  • EXAMPLE 1 Production of conditioned mean from culture of mesenchymal cells of Warthon's jelly from umbilical cord.
  • the conditioned mean was obtained from following procedure:
  • a dermal equivalent was produced by the following protocol:
  • a cutaneous equivalent was produced by the following procedure.
  • EXAMPLE 4 Use from dermal equivalent in patients with ulcers.
  • the dermal equivalent obtained was used in the treatment of ulcers, in accordance with the following procedure.
  • EXAMPLE 5 Use from cutaneous equivalent in patients with burns from autologous cells.
  • the cutaneous equivalent obtained according with the present invention was used in the treatment from patients with burns, according with the following procedure.
  • Table 1 the general data from patients participating in the experiment are shown including gender, age, evolution, consumption of NSAIDs, comorbidities and size from wound to each of the two compared groups.
  • a statistical comparison from mean values is included to each considered parameter in accordance with the statistic test applied, a P value greater than 0.05 indicate the existence of statistically significant differences between both groups.
  • a P value greater than 0.05 indicate the existence of statistically significant differences between both groups.
  • statistically significant differences do not exist between both groups for the measured parameters.
  • Table 3 and Figures 4 and 5 a comparison it shows between the means from area of ulcer, from area from ulcer in the eight week, the mean reduction, the area from reduced ulcer of, the percentage from closed ulcer, the closing velocity and percentage of patients with total closing at 9 weeks of treatment for each from two groups compared. Same in the above tables, a statistic comparison from mean values is included to each considered parameter. As shown in the P column from Table 3, statistically significant differences do not exist in any of measured parameters between the treatments of ulcers using the dermal equivalent generated using the supplement elaborated by the developed method and a conventional hydrocolloid dermal equivalent. Although statistically significant there is no decrease in the area from ulcers between both treatments, there is a significant reduction from approximately twice the size from wounds in the group of patients treated with the dermic equivalent.
  • the average weekly cost is presented from treatment using dermal equivalents available commercially and the cost of dermal equivalent elaborated using the supplement from present invention.
  • the average cost from weekly treatment using the developed dermal equivalent is widely less to the average cost from conventional treatments; at the same time maintain the therapeutic activity.
  • the treatment with Oasis is of lower price; however, does not have autologous cells such as the case from described in the developed dermal equivalent in accordance with the method described in the present invention.
  • Dermalogen is an acellular collagen matrix obtained from skin of cadaveric donators and is equal in costs to dermal equivalent described in accordance to the developed method in the present invention, On the other hand, its manufacture method is totally different to dermal equivalent described here. Table 4.

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Claims (8)

  1. Verfahren zur Herstellung eines Supplements zur Begünstigung der Invitro-Zellproliferation von Zellen des kutanen Systems, umfassend die folgenden Schritte:
    a) Kultivieren von mesenchymalen Zellen von Wharton-Sulze in einem geeigneten Kulturmedium mit Entwicklungsfaktoren, um Konfluenz zu erreichen, wobei die Entwicklungsfaktoren epidermaler Wachstumsfaktor (EGF) und basischer Fibroblasten-Wachstumsfaktor (bFGF) sind;
    b) Gewinnen der kultivierten Zellen aus Schritt a) und weiteres Kultivieren der Zellen in DMEM 50 % - F12 50 % mit fötalem Rinderserum mit Antibiotika, ohne Entwicklungsfaktoren, um ein konditioniertes Medium zu erhalten;
    c) Sammeln des konditionierten Mediums aus Schritt b);
    d) Wiederholung der Schritte a) und b) mindestens einmal, um ein angereichertes konditioniertes Medium zu erhalten; und
    e) Konzentrieren des angereicherten konditionierten Mediums aus Schritt d) und Sterilisieren durch Filtration, um das Supplement zu erhalten.
  2. Verfahren zur Begünstigung der Entwicklung und Aktivierung von Zellen des kutanen Systems in vitro, umfassend das Kultivieren der Zellen in einem Kulturmedium, das das nach Anspruch 1 hergestellte Supplement enthält.
  3. Verfahren nach Anspruch 2, dadurch gekennzeichnet, dass die Zellen des kutanen Systems Fibroblasten, Keratinozyten, dendritische Zellen, Monozyten oder Kombinationen davon umfassen.
  4. Verfahren zur Herstellung eines dermalen Äquivalents, umfassend die Schritte:
    - Kultivieren autologer oder allogener Fibroblasten in einem Kulturmedium, das den nach Anspruch 1 hergestellten Zusatz enthält, um eine geeignete Konfluenz zu erhalten;
    - Gewinnen durch enzymatische Behandlung und Waschen der im vorhergehenden Schritt erhaltenen Fibroblasten;
    - Wiedereinsäen der im vorhergehenden Schritt erhaltenen Fibroblasten in ein Kulturmedium, das das nach Anspruch 1 hergestellte Supplement in einer Supplementkonzentration von 5 - 50 % in Bezug auf das Gesamtvolumen des Kulturmediums enthält, und Wachsen bis zu einer geeigneten Konfluenz; und
    - Erzeugen des dermalen Äquivalents.
  5. Verfahren zur Herstellung eines kutanen Äquivalents, umfassend die Schritte:
    a) Erhalten eines dermalen Äquivalents durch das Verfahren nach Anspruch 4;
    b) Erhalten von Keratinozyten aus einem autologen oder allogenen System;
    c) Kultivieren der Keratinozyten aus Schritt b) in einem Kulturmedium, das den nach Anspruch 1 hergestellten Zusatz enthält, um eine geeignete Konfluenz zu erhalten;
    d) Gewinnen durch enzymatische Behandlung und Waschen der in Schritt c) erhaltenen Keratinozyten;
    e) Wiedereinsäen der in Schritt d) erhaltenen Keratinozyten in ein Kulturmedium, das das nach Anspruch 1 hergestellte Supplement in einer Supplementkonzentration von 5 - 50 % in Bezug auf das Gesamtvolumen des Kulturmediums enthält, und Wachsen bis zu einer geeigneten Konfluenz; und
    f) Kultivieren der in Schritt 3) erhaltenen Keratinozyten über dem dermalen Äquivalent von Schritt a), um das kutane Äquivalent zu erzeugen.
  6. Verfahren nach Anspruch 4, dadurch gekennzeichnet, dass die Fibroblasten aus einem autologen oder allogenen System menschlichen oder tierischen Ursprungs stammen.
  7. Verfahren nach Anspruch 5, dadurch gekennzeichnet, dass die Keratinozyten aus einem autologen oder allogenen System menschlichen oder tierischen Ursprungs stammen.
  8. Verfahren zur Herstellung einer Zellsuspension von Fibroblasten, umfassend die folgenden Schritte:
    a) Kultivieren von Fibroblasten in einem Kulturmedium, das das nach Anspruch 1 hergestellte Supplement umfasst, um eine geeignete zelluläre Konfluenz zu erreichen;
    b) Abtrennen durch eine enzymatische Behandlung und Waschen der in Schritt a) erhaltenen Fibroblasten vom Kulturmedium;
    c) Wiedereinsäen der in Schritt b) erhaltenen Fibroblasten in ein Kulturmedium, das das nach Anspruch 1 hergestellte Supplement in einer Supplementkonzentration von 5 - 50 % in Bezug auf das Gesamtvolumen des Kulturmediums enthält, und Wachsen bis zu einer geeigneten Konfluenz; und
    d) Einbringen der in Schritt c) erhaltenen Fibroblasten in eine Lösung aus autologem Blutserum.
EP17853510.0A 2016-09-23 2017-09-25 Verfahren zur herstellung eines supplements aus mesenchymalen zellkulturen von wharton-sulze und verwendungen davon Active EP3517606B1 (de)

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